This application is a request for funds to continue studies on the role of adenine nucleotides and nucleosides in sympathetic neuroeffector process. The evidence is substantial that ATP plays an important role, along with norepinephrine (NE), as a sympathetic nerve cotransmitter and comodulator. The plan has three specific aims. The first specific aim is to examine the hypothesis that prejunctional alpha 2-adrenoceptors function as sensors that "read" the frequency of action potentials produced during bursts of neuronal activity and convert that information into discrete chemical messages with varying proportions of cotransmitters. These experiments are intended to extend our knowledge about the receptor mechanisms involved in nerve activity-dependent regulation of the ratio of ATP and NE released from sympathetic nerves. Our preliminary results show that there is a causal relationship between the frequency of nerve stimulation and the amount of NE that is released together with relatively constant amounts of ATP. The changes in composition of the cotransmitter cocktail and the amplitude of the smooth muscle contractile response appear to be regulated by the prejunctional alpha 2-adrenoceptors, which withhold NE from the cotransmitter mixture at low but not at high frequencies of nerve stimulation. To further confirm this concept we propose to carry out experiments with vascular and non-vascular neuroeffector preparations deficient in alpha 2-adrenoceptors. The second specific aim is to examine the hypothesis that by releasing soluble nucleotidases the sympathetic nerves contribute to both clearance of neurotransmitter ATP, and regulation of the level of extracellular adenosine (ADO). An extension of this hypothesis is that ADO produced as a result of the accelerated metabolism of extracellular ATP during nerve stimulation modulates neurotransmitter release. The rationale for this study is based on our newly developed evidence, supported by HL 38126, demonstrating that stimulation of sympathetic nerves releases soluble nucleotidases. We plan to correlate the changes in amplitude and time-course of nerve stimulation-evoked release of NE from the guinea pig vas deferens with the time course of production of ADP, AMP and ADO resulting from the breakdown of exogenously applied ATP and slowly degradable analogues of ATP. The third specific aim is to isolate and determine the protein structure of the soluble nucleotidases released during stimulation of sympathetic nerves. Standard protein isolation methods will be employed.